HIV-drug susceptibility is associated with virologic response to new treatments. Standardized drug resistance tests are now available, and data from clinical trials suggest that the use of drug resistance testing may be associated with improved virologic outcome. However, drug resistance is complex in terms of performance, interpretation and clinical application.
Phenotypic tests measure the ability of an HIV isolate to grow in the presence of drug and is performed using assays in which the degree of virus replication inhibition at different drug concentrations is assessed. Results are used to calculate the 50% or 90% inhibitory concentration of a drug for an isolate. A potential problem with the classic phenotype assay is the effect of genetic drift in the virus population during virus isolation. In worst case the virus clone isolated is the one most fit to grow during in vitro conditions, not the one most abundant in the patient.
Measurement of phenotypic drug susceptibility can now be done via automated assays based on recombinant DNA technology. These approaches involve amplification of plasma RNA coding for HIV protease and reverse transcriptase (RT) and generation of a recombinant virus with the other genes from a laboratory construct (cassette virus) [1]. Genotype assays measure the occurrence of certain mutations in the genes targeted by antiretroviral drugs. Whereas phenotypic resistance measures virus-drug susceptibility, genotyping detects the mutations that confer phenotypic resistance. Polymerase chain reaction (PCR) amplification of HIV-1 sequences from plasma containing 500 to 1000 RNA copies per ml is the initial step in both these assays and in the recombinant virus phenotypic assays. Depending on mutations assessed and laboratory performing, the test, genotype assays may differentiate a mutant at a level of 10 to 50% in a mixture of viruses. The complexity of the data generated from sequencing has led to difficulties in interpreting the results. There may be varying interpretations regarding the level of phenotypic resistance conferred by a specific mutational pattern. As new data are generated, there is a risk of providing inadequate or even incorrect interpretations [2]. The reaction mechanism for all antiretroviral drugs used hitherto is in interference with the enzymatic reaction of either the viral protease or the RT. Depending on the capacity of the enzyme assays and the virus isolation techniques used, the drug sensitivity testing can theoretically be done either on supernatants from virus culture propagation, the primary virus isolation or on virus preparations recovered directly from the patients.
Drug susceptibility testing on RT from the primary virus isolation offers two advantages compared to traditional virus replication inhibition tests. The time for virus propagation is reduced and more important, less selection on the virus population will occur. The ideal situation is finally to characterize enzyme which has been extracted directly from the virus circulating in the blood of the patient. The benefit of such an approach is that the sample will mirror the virus population present in the patient at the time the blood sample was taken. This has so far not been feasible in practice, but the concept has been explored by resistance testing with the PCR based Amp RT assay [3].
The HIV-1 RT as well as other reverse transcriptases perform three different enzymatic reactions: RNA-dependent DNA polymerization, DNA-dependent DNA polymerization, and degradation of RNA in the DNA-RNA hybrid (RNase H). The HIV RT, encoded by the pol gene, is a heterodimer consisting of a p66 and a p51 subunit. Both RNA-dependent DNA polymerization and DNA-dependent DNA polymerization are performed by the same active site localized in the p66 subunit. The p51 is produced by removal of the C-terminal fragment of the p66 corresponding to the RNase H domain [4]. All RT inhibitors currently approved for clinical use inhibit the polymerase activity of the enzyme. The reaction mechanism of these drugs has mainly been defined according to their action on the RNA-dependent DNA polymerization reaction. The effect on the DNA-dependent DNA polymerization reaction is comparatively less studied.
Conventional RT activity assay is performed by utilising an artificial template-primer construction and labelled deoxynucleotide triphosphate as nucleotide substrate. The template/primer pair poly(rA)/oligo(dT) is the most efficient and most used combination for determination of HIV as well as for other retroviral RTs. A drawback of this type of assay when drug sensitivity testing is concerned, is that only non-nucleoside analogues or analogues that can base pair with rA can be tested. Analogues to the other nucleotide bases will require an assay based on a variable polymer template.
All anti-retroviral drugs approved hitherto interfere with the enzymatic reaction of either the viral protease or the RT. There are in addition candidate drugs in the pipeline which affect the function of the retroviral integrase.
The RT inhibitors are either nucleoside analogues or non-nucleoside analogues. The non-nucleoside inhibitors bind to a hydrophobic pocket in the RT enzyme close to, but not contiguous with the active site. HIV-1 replication is inhibited allosterically by displacing the catalytic aspartate residues relative to the polymerase binding site. Resistance usually emerges rapidly when non-nucleosides are administered as monotherapy or in the presence of incomplete virus suppression. Currently only three non-nucleoside inhibitors: Nevirapine, Efavirenz and Delavirdine are approved for clinical use by FDA.
The nucleoside inhibitors used today terminate the DNA chain elongation as they lack a 3′-hydroxyl group. Prolonged therapy with nucleoside inhibitors commonly leads to the development of resistant virus. This process is associated with the gradual appearance of mutations in the virus pol gene, each leading to defined amino acid substitutions [5]. The effects of these substitutions at the enzymatic levels are complicated and includes enhancement of a primitive DNA editing function. This reaction is nucleotide dependent and produces dinucleoside polyphosphate and an extendible DNA 3′end [6].
HIV therapy today is based on multidrug therapy. The regimens are based on combinations of all three types of drugs available: nucleoside analogues, non-nucleoside analogues and protease inhibitors. The strategy is to minimise the probability for a mutant virus to survive. Facing virologic failure current therapy guidelines recommend switch to an entirely new batch of drugs. This is frustrating since many HIV positive people do not have three or more untried drugs from which to choose. Further it may also be a wasteful decision to remove a drug that in fact still is effective. With improved drug resistance testing it might be possible to weed out the ineffective drug or drugs in a given combination.